Stimulation of the steady state ATPase kinetics of the ABC exporter NaAtm1 by a transported substrate GSSG

FOR THE RECORD

Modeling the stimulation by glutathione of the steady state

kinetics of an adenosine triphosphate binding cassette

transporter

Chengcheng Fan

| Douglas C. Rees

Division of Chemistry and Chemical

Engineering, Howard Hughes Medical

Institute, MC 114-96, California Institute

of Technology, Pasadena, California, USA

Correspondence

Douglas C. Rees, 1200 E. California Blvd.,

MC 114-96, Pasadena, CA 91125, USA.

Email: dcrees@caltech.edu

Present address

Chengcheng Fan, Division of Biology and

Biological Engineering, MC 114-96,

California Institute of Technology,

Pasadena, California, USA

Funding information

Howard Hughes Medical Institute

Abstract

We report the steady state ATPase activities of the ATP Binding Cassette

(ABC) exporter

Atm1 in the absence and presence of a transported sub-

strate, oxidized glutathione (GSSG), in detergent, nanodiscs, and

proteoliposomes. The steady state kinetic data were fit to the

“

nonessential

activator model

”

where the basal ATPase rate of the transporter is stimulated

by GSSG. The detailed kinetic parameters varied between the different recon-

stitution conditions, highlighting the importance of the lipid environment for

Atm1 function. The increased ATPase rates in the presence of GSSG more

than compensate for the modest negative cooperativity observed between

MgATP and GSSG in lipid environments. These studies highlight the central

role of the elusive ternary complex in accelerating the ATPase rate that is at

the heart of coupling mechanism between substrate transport and ATP

hydrolysis.

KEYWORDS

ABC transporter, ATPase activity, glutathione, Michaelis

–

Menten kinetics, nonessential

activator model

INTRODUCTION

The ability of substrates to stimulate the ATPase activity

of ATP binding cassette (ABC) transporters has been uti-

lized to identify potential substrates transported by mem-

bers of this ubiquitous family.

–

Despite the practical

significance of this approach for identifying the sub-

strates of ABC exporters, more detailed kinetic character-

izations of the ATPase activity as a function of the

concentrations of both MgATP and transported substrate

have not, to our knowledge, been reported. Such mea-

surements could provide insight into the coupling

between substrate binding and ATPase activity that is

central to the mechanism of substrate translocation

by ABC transporters.

–

We accordingly conducted

such studies for the bacterial ABC exporter from

Novosphingobium aromaticivorans

(

Atm1), a homolog

of the ABC transporter of mitochondria 1 (Atm1) family

of ABC exporters.

–

We have previously established that oxidized glutathi-

one (GSSG) can be transported by

Atm1.

Together

with structural analyses that have captured multiple con-

formational states, a framework for an alternating access

transport cycle has been defined, with the interconver-

sion between states coupled to the binding and hydrolysis

of ATP.

13,14

These studies have established that ATP or

Received: 6 October 2021

Revised: 3 December 2021

Accepted: 3 December 2021

DOI: 10.1002/pro.4250

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any me

dium, provided

the original work is properly cited.

Protein Science

published by Wiley Periodicals LLC on behalf of The Protein Society.

752

Protein Science.

2022;31:752

–

757.

wileyonlinelibrary.com/journal/pro

related nucleotides are required for the formation of

outward-facing conformations by

Atm1. In contrast,

GSSG has only been found associated with the inward-

facing conformational state. MgATP hydrolysis requires

the formation of a closed dimer interface between the

two nucleotide binding domains (NBDs), with two nucle-

otides sandwiched between catalytic groups contributed

by both domains.

The catalytically relevant juxtaposi-

tion of dimerized-NBDs is associated with the outward-

facing conformation. The preferential binding of GSSG to

the inward-facing conformation while the ATPase activ-

ity requires the outward-facing conformation poses a par-

adox since it would seemingly predict that the

transported substrate should inhibit the ATPase activity

rather than stimulate it, as is observed. This prediction

reflects the expectation that the transported substrate

should thermodynamically stabilize the inward-facing

conformation that is expected to be the ATPase inactive

state. As a first step to resolving this paradox, we charac-

terized the dependence of the steady state ATPase kinet-

ics on the concentrations of MgATP and the transported

substrate, oxidized glutathione (GSSG), for the ABC

exporter

Atm1.

RESULTS AND DISCUSSION

2.1

ATPase activities

The dependence of the ATPase activity on the concentra-

tion of GSSG was measured for

Atm1 reconstituted

under three different conditions:

•

a detergent mixture composed of 0.05% DDM and

0.05% C12E8 used in the crystallization studies.

13,14

•

nanodiscs composed of the membrane scaffolding pro-

tein MSP1D1 and the phospholipid 1-palmitoyl-2-

oleoyl-glycero-3-phosphocholine (POPC).

•

proteoliposomes composed of

E.coli

polar lipids and

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

The ATPase rates were measur

ed in sextuplicate for the

detergent and proteoliposom

e samples, and triplicate for

the nanodisc sample under varying concentrations of

MgATP (8 concentrations: 0, 0.1, 0.2, 0.5, 1, 2, 5, and

10mM)andGSSG(6concentrations,0,1,2.5,5,10,and

20 mM). The ATPase rate was measured by quantifying the

amount of inorganic phosphate (Pi) released upon ATP

hydrolysis over a 15-min period using a molybdate based

colorimetric assay.

For a given GSSG concentration, the

dependence of the ATPase activity on (MgATP) was

modeled by a Michaelis

–

Menten (hyperbolic) equation

(Figure 1), yielding

cat

andKmforATPhydrolysisasa

function of GSSG concentration (Table S1). In the absence

of GSSG, the values of

cat

characterizing the basal

(uncoupled) ATPase rate were determined to be 18.0 ± 0.4,

FIGURE 1

Fit of the ATPase activities of

Atm1 to the

Michaelis

–

Menten kinetic model. ATPase activities of

Atm1 as a

function of (MgATP) in (a) detergent (DDM/C12E8), (b) nanodiscs,

and (c) proteoliposomes, with stimulations by GSSG at various

concentrations. ATPase activities of

Atm1 in detergent and PLS

were measured six times, and three times in nanodiscs, all with

distinct samples. All of the extrapolated

max

values are shown next

to each GSSG concentration curve. Error bars represent the

standard error of the mean for the replicates

FAN

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753

30.9 ± 0.7, and 9.5 ± 0.4 min

in detergent, nanodiscs,

and proteoliposomes, respectively. As the orientation effect

in proteoliposomes was not taken into account, the actual

ATPase rate in proteoliposomes may be ~2 times of the

measured activity, or ~19 min

.Ineachsystem,GSSGwas

observed to stimulate the ATPase activity of

Atm1 in a

concentration dependent fashion (Figure 1 and Table S1).

The magnitude of the stimul

ationwasdependentonthe

reconstitution conditions, as the

cat

s in 20 mM GSSG were

increased above the basal ra

te in the absence of GSSG by

factors of ~5, 14, and 11 (Table S1a), in detergent,

nanodiscs, and proteoliposom

es, respectively. Less pro-

nounced, but statistically si

gnificant changes in Km were

also observed between 0 and 2

0mMGSSG,corresponding

to a decrease of ~20% in deterg

ent, and increases of 2.7

times and 1.9 times for nanodiscs and proteoliposomes,

respectively (Table S1b).

2.2

Basic nonessential activator kinetic

model

To model this data, we used a nonessential activator

model (Figure 2a), a steady-state, equilibrium binding

model where the transpo

rted substrate GSSG is an

activator that stimulates the ATPase rate above the

basal level.

19,20

The key kinetic parameters in this

model are:

, the Michaelis binding constant for MgATP,

ii.

, the Michaelis binding constant for the trans-

ported substrate, GSSG, which is also an activator of

the ATPase rate,

iii.

, the interaction factor for how binding of MgATP

influences the binding of GSSG (and vice versa);

< 1 or > 1 denote positive and negative coopera-

tivity, respectively,

iv.

, the basal rate constant for MgATP hydrolysis in

the absence of GSSG, and

, the acceleration factor for MgATP hydrolysis with

bound GSSG.

In this basic model, the ATPase sites are treated as inde-

pendent since the dependence of the ATPase rate on ATP

is reasonably well approximated by the hyperbolic

Michaelis

–

Menten equation, except possibly at the lowest

concentrations of ATP where some evidence for coopera-

tivity was observed. For this scheme, expressions for the

overall velocity,

obs

and

app

, may be derived (where

denotes the total concentration of transporter).

FIGURE 2

Nonessential activator model of

Atm1 ATPase kinetics. (a) Schematic of the nonessential activator kinetic model for the

ATPase activities of

Atm1. Fits of (b) the apparent rate constant,

obs

, for ATP hydrolysis (Equation (2)) and (c) the Michaelis

–

Menten

constant,

, of MgATP binding (Equation (3)) as a function of (GSSG) based on the experimentally derived parameters (Table 1) for the

nonessential activator model for

Atm1 in detergent, nanodics, and proteoliposomes, respectively. In these schemes,

=

Atm1,

=

MgATP,

=

ADP,

=

GSSG,

=

binding constant for MgATP,

=

binding constants for GSSG,

=

rate constant for MgATP

hydrolysis,

=

interaction factor of how ATP binding influences GSSG binding and vice versa, and

=

acceleration factor for ATP

hydrolysis with bound GSSG, Error bars represent the experimentally observed standard deviations

754

FAN

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:

obs

:

app

:

The parameters (

, and

) of the nonessential

activator model (Equation (1), Table 1) were fit against

the 48 measured ATPase rates as a function of ATP and

GSSG concentrations in detergent, nanodiscs, and

proteoliposomes. The basal turnover rates observed for

saturating MgATP in the absence of GSSG, k, were deter-

mined to be 17.6 min

in detergent, 32 min

nanodiscs and 9 min

in proteoliposomes (Table 1), with

the binding affinities of MgATP,

), measured as

0.82 mM in detergent, 1.41 mM in nanodiscs and 1.6 mM

in proteoliposomes. The determined values for K

, the

binding constant of GSSG, were found to be ~10 mM

under all reconstitution conditions. Extrapolating to satu-

rating concentrations of GSSG, the acceleration factors

were determined to be 8.3, 77, and 29 in detergent,

nanodiscs, and proteoliposomes, respectively (Table 1).

With these values for the parameters of the nonessential

activator model, the experimental values of

cat

and Km

as a function of (GSSG) were fit reasonably well

(Figure 2bc), as were the fit of the individual ATPase

measures as a function of MgATP and GSSG concentra-

tions (Figure S1).

The results of this analysis demonstrate the ATPase

kinetics are dependent on the lipid environment,

explored in this work as detergent, nanodiscs, and

proteoliposomes, and that the basal ATPase rates are

stimulated by GSSG under these reconstitution condi-

tions. While the primary influence of GSSG is to acceler-

ate

cat

, the Km values for ATP are also impacted. These

results provide the opportunity to explore the binding

interactions between ATP and GSSG, as reflected in the

cooperativity parameter

. While little cooperativity is

evident in detergent (

~ 1.025), in the lipid environment

provided by reconstitution into nanodiscs and

proteoliposomes, evidence for modest negative coopera-

tivity is observed with

~ 10 and 3, respectively. These

trends are similarly reflected in the increases in

, the

Michaelis constant for ATP, between 0 and 20 mM

observed for nanodiscs and proteoliposomes.

For an allosteric system described by a classical

Monod

–

Wyman

–

Changeux model,

a ligand that prefer-

entially binds to the inactive conformation of a two-state

system will function as an inhibitor. GSSG appears to

bind preferentially to the inward-facing conformation of

Atm1, while the catalytically competent conformation

for ATP hydrolysis is the outward-facing conformation.

Based on an equilibrium binding model, it would be

anticipated that GSSG and MgATP should exhibit nega-

tive cooperativity towards each other. This expectation is

reflected in the

> 1 values for

Atm1 reconstituted in

a lipid environment, corresponding to the increases in

between 0 and 20 mM GSSG under those conditions. Nev-

ertheless, GSSG significantly stimulates the ATPase rate,

which suggests that the kinetics of forming the outward-

facing conformations of

Atm1 differ in important ways

between the binary (with MgATP) and ternary (with

MgATP and GSSG) complexes. A key mechanistic ques-

tion is why the ternary complex has an accelerated

ATPase rate. Structural studies have yet to provide any

insights into this question as no structures of this ternary

complex have been determined for an ABC exporter.

Understanding how this species promotes ATP hydrolysis

relative to the binary complex is at the heart of the cou-

pling mechanism and emphasizes the importance of char-

acterizing the structure and dynamics of this elusive

transporter-ATP-substrate ternary state.

MATERIAL AND METHODS

3.1

Protein expression, purification,

and reconstitution

The over-expression of

Atm1 (Addgene catalog num-

ber 78308) was achieved with

Escherichia coli

BL21-gold

TABLE 1

Kinetic parameters of the nonessential activator

model

Parameters Detergent Nanodiscs Proteoliposomes

obs

(min

) 17.58 ± 0.75 31.86 ± 1.04 9.49 ± 1.26

(mM)

0.82 ± 0.07 1.41 ± 0.08 1.64 ± 0.34

(mM)

13.34 ± 2.20 9.65 ± 0.74 12.79 ± 4.09

lpha (

)

1.03 ± 0.20 10.05 ± 1.73 2.64 ± 1.26

Beta (

)

8.30 ± 0.46 76.60 ± 8.68 28.69 ± 6.30

Note

: Parameters calculated with the nonessential activator model shown in

Figure 2 for the ATPase activities of

Atm1 in detergent, nanodiscs, and

proteoliposomes. The

values are in the range of 0.94 to 0.99 for the

measurements in detergent, 0.99

–

1.00 for the measurements in nanodiscs

and 0.91

–

0.95 for the measurements in proteoliposomes. Parameters are

tabulated to two decimal places to accurately reproduce the calculations

depicted in Figures 2 and S1.

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(DE3) cells (Agilent Technologies) using ZYM-5052 auto-

induction media as described previously.

Cells were

harvested by centrifugation and stored at

until use.

Protein purification was carried out as previously

described.

Briefly, frozen cell pellets were resuspended

in lysis buffer containing 100 mM NaCl, 20 mM Tris,

pH 7.5, 40 mM imidazole, pH 7.5, 10 mM MgCl

, 0.5%

(wt/vol)

n-dodecyl-

-maltopyranoside

(DDM)

(Anatrace), and 0.5% (wt/vol) octaethylene glycol mono-

dodecyl ether (C12E8) (Anatrace) in the presence of lyso-

zyme, DNase, and protease inhibitor tablet. Resuspended

cells were solubilized by stirring for 3 h at 4

C. The lysate

was ultracentrifuged at 113,000 x

for 45 min at 4

Cto

remove unlysed cells and cell debris. The supernatant

was loaded onto a prewashed NiNTA column. NiNTA

wash buffer contained 100 mM NaCl, 20 mM Tris,

pH 7.5, 50 mM imidazole, pH 7.5, 0.05% DDM and 0.05%

C12E8 and elution buffer contains 350 mM imidazole

instead. The eluted sample was then subjected to size

exclusion chromatography using HiLoad 16/60 Superdex

200 (GE Healthcare) with buffer containing 100 mM

NaCl, 20 mM Tris, pH 7.5, 0.05% DDM, and 0.05%

C12E8. Peak fractions were collected and concentrated

with Amicon Ultra 15 concentrator (Millipore)

(MW 100 kDa) to ~20 mg/ml.

Atm1 nanodiscs in membrane scaffolding protein

and proteoliposomes were prepared as described previ-

ously.

Additional BioBeads were added at 50 mg/ml to

ensure the complete removed of detergent in both recon-

stitutions. The protein concentration in the nanodisc

preparations includes contributions from both the trans-

porter and the scaffold protein; for calculating the

cat

values, it is assumed that the ratio of scaffold protein to

transporter in the nanodiscs is 2:1 based on the EM den-

sity. Consequently, the transporter concentration was cal-

culated as 135/(135

+

25)

=

0.73 of the total protein

concentration based on molecular weights of 25 kDa and

135 kDa for the scaffold protein and

Atm1,

respectively.

3.2

ATPase assay

The ATPase activities were measured by the molybdate

based phosphate quantification method

as described

previously.

All reactions were carried out at 37

Cin

250

l scale. The reaction mixture contained a final

Atm1 concentration of 0.05 mg/ml in 100 mM NaCl

and 20 mM Tris, pH 7.5, with varying concentrations of

MgATP and GSSG. For each reaction, 50

l of reaction

mixture was taken every 5 min for 4 times and subse-

quently mixed with 50

l of 12% SDS, 100

l of ascorbic

acid/molybdate mix, and 150

l of citric acid/arsenite/

acetic acid solution before reading with a Tecan plate

reader at 850 nm. Reactions in detergent and

proteoliposomes were done in sextuplicates and reactions

in nanodiscs were done in triplicates. The measurements

were plotted against time to obtain the ATPase rates. The

final rates were fitted into Michaelis

–

Menten kinetics or

the nonessential activator model in Mathematica and

Prism 9. The graphs were plotted in Prism 9. The

unweighted least regression resulted in

values of 0.98,

1.00, and 0.95 for the measurements in detergent,

nanodiscs, and proteoliposomes, respectively.

CONFLICT OF INTEREST

The authors declare no competing interests.

AUTHOR CONTRIBUTIONS

Chengcheng Fan:

Conceptualization (equal); data

curation (lead); formal analysis (equal); investigation

(lead); methodology (lead); validation (lead); visualization

(lead); writing

–

original draft (lead); writing

–

review and

editing (equal).

Douglas C. Rees:

Conceptualization

(equal); formal analysis (equal); funding acquisition (lead);

project administration (lead);

supervision (lead); writing

–

review and editing (equal).

ORCID

Chengcheng Fan

https://orcid.org/0000-0003-4213-5758

Douglas C. Rees

https://orcid.org/0000-0003-4073-1185

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SUPPORTING INFORMATION

Additional supporting information may be found in the

online version of the article at the publisher's website.

How to cite this article:

Fan C, Rees DC.

Modeling the stimulation by glutathione of the

steady state kinetics of an adenosine triphosphate

binding cassette transporter. Protein Science. 2022;

31:752

–

https://doi.org/10.1002/pro.4250

FAN

AND

REES

757